The present invention relates to recombinant DNA technology, and in particular to introducing foreign nucleic acid in a eukaryotic cell, and more particularly to compositions and methods for producing alphavirus replicon particles useful in immunotherapies and/or gene therapy applications. In particular, the present invention discloses a genetic background for the alphavirus replicon particle system that is based on the Venezuelan Equine Encephalitis virus (VEE) vaccine strain, TC-83.
A variety of viruses is included in the alphavirus genus, which is a member of the Togaviridae family. The alphaviral genome is a single-stranded, messenger-sense RNA, modified at the 5′-end with a methylated cap and at the 3′-end with a variable-length poly (A) tract. Structural subunits containing a single viral protein, capsid, associate with the RNA genome in an icosahedral nucleocapsid. In the virion, the nucleocapsid is surrounded by a lipid envelope covered with a regular array of transmembrane protein spikes, each of which consists of three heterodimeric complexes of two glycoproteins, E1 and E2. See Paredes et al., (1993) Proc. Natl. Acad. Sci. USA 90:9095-9099. The Sindbis and Semliki Forest viruses are considered the prototypical alphaviruses and have been studied extensively. See Schlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986). The VEE virus has also been studied extensively, see, e.g., U.S. Pat. Nos. 5,185,440, 5,505,947, and 5,643,736.
The use of propagation-defective alphavirus particles, termed alphaviral replicon particles, has shown great promise as a viral vector delivery system. Replicons are constructed to carry one or more heterologous antigens in place of some or all of the alphavirus structural genes. The replicons are introduced into alphavirus-permissive cells along with a helper construct(s) that expresses the viral structural protein(s) not encoded by the replicon or, alternatively, the replicon is introduced into a packaging cell capable of expressing the structural proteins. The replicon is then packaged, analogous to the packaging of the intact alphaviral genome, by the expressed structural proteins. These packaged replicons, or alphaviral replicon particles, are then inoculated into an animal. The particles enter the host cell, and the replicons then express the introduced heterologous coding or other functional sequence(s) at very high levels from the subgenomic mRNA. Subsequent viral progeny are prevented from assembly since the replicons do not encode all of the essential viral packaging (structural) genes.
Both the alphaviral genetic background for the replicon and the alphaviral structural proteins used to package the replicon have a significant impact on the ultimate performance of the replicon particles. The VEE virus has been preferred as a vaccine vector among the alphaviruses because it is naturally lymphotrophic, which leads to strong cellular and humoral immune responses at relatively low immunization doses (Davis, N L et al. (1996) J. Virol. 70(6): 3781-7; MacDonald, G H and Johnston R E, (2000) J. Virol. 74(2): 914-922; Caley I J et al. (1997) J. Virol. 71: 3031-3038; Hevey M et al. (1998) Virology 251(1): 28-37; Caley I J et al. (1999) Vaccine 17:23-24; Pushko, P et al. (2001) Vaccine 19:142-153).
Several strains of the Venezuelan Equine Encephalitis virus (VEE) are known, and within those strains, subtypes have been recognized. Virulent VEE strains have been isolated during mosquito-borne epidemic encephalomyelitis in equids in tropical and sub-tropical areas of the New World. One of the most virulent epizootic strains, the Trinidad Donkey (TRD) strain, which is in subtype IA/B, was passaged serially in tissue culture to create a live, attenuated strain (Berge et al. (1961) Amer. J. Hyg. 73:209-218) known as TC-83. This strain elicits VEE-specific neutralizing antibodies in most humans and equines and has been used successfully as a vaccine in both species (McKinney et al. (1972) “Inactivated and live VEE vaccines—A Review, in Venezuelan Encephalitis, pp. 369-376, Sc. Pub. No. 243 Pan American Health Organization, Washington, D.C.; Walton T E et al. (1972) Am. J. Epidemiol. 95:247-254; Pittman P R et al. (1996) Vaccine 14(4): 337-343). Nonetheless, this vaccine presents several problems in terms of safety and efficacy. First, it can cause adverse, sometimes moderately severe reactions in human vaccines. Second, the TC-83 strain shows residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation (Ludwig G et al. (2001) Am. J. Trop. Med. Hyg. January-February; 64(1-2):49-55). Third, the TC-83 strain has a significant percentage of non-responders in humans, i.e., individuals who do not show a demonstrable humoral response after inoculation (Pittman P R et al. (1996) Vaccine 14(4): 337-343). Finally, the TC-83 strain is known to be especially sensitive to interferon, as compared to the parental TRD strain or other epizootic strains of VEE (Spotts, D R et al. (1998) J. Virol. 72:10286-10291). Such enhanced sensitivity to interferon would lead one to expect that the heterologous genes in a replicon particle would be expressed less efficiently in an infected cell and/or that such particles would be less immunogenic in vivo. All of these detrimental factors associated with the TC-83 vaccine strain of VEE have led previous researchers to search for better attenuated strains to use as either propagation-competent VEE vectors or in replicon particle systems (e.g. Davis N L et al. (1994) Arch. Virol. Suppl. 9:99-109; Davis N L et al. (1996) J. Virol. 70(6):3781; Pushko et al. (1997) Ibid.; Pratt W D et al. (2003) Vaccine 21(25-26): 3854-3862).
There is a continuing need to optimize the combination of mutations and alphavirus strain to provide the most effective alphavirus replicon particle for use in vaccine and/or gene therapy applications.